The galactose:phosphotransferase and the lactose:phosphotransferase systems have been characterized in Lactobacillus casei. An intracellular hexose-6-phosphate specific phosphohydrolase has been purified from Streptococcus lactis. High resolution 31P-nuclear magnetic resonance techniques have been used to monitor in vivo concentrations of inorganic phosphate (Pi), and phosphorylated intermediates in glycolysing and starved cells of Streptococcus lactis. These experiments have demonstrated the importance of Pi in the modulation of pyruvate kinase activity. Lactose derivatives containing [14C] 2-deoxyglucose (2DG) of [14C] 2-fluoroglucose (2FG) as aglycon substituents of the disaccharide have been synthesized enzymatically. These compounds serve as biochemical Trojan horses and permit cells (which would normally exclude 2DG and 2FG) to be preloaded with the non-metabolizable hexose analogs. The lactose analogs serve as probes for the route of 2DG and 2FG efflux from mannose-PTS defective mutants. The three stages of an energetically wasteful 2DG-futile cylce in S. lactis have been elucidated. Evidence has been obtained for the futile cycling of non-metabolizable pentitols in L. casei.